Applications of Chromatography

Applications of Chromatography 1

APPLICATIONS OF CHROMATOGRAPHY

Chromatography is a process used to separate mixtures. The word chromatography is derived

from the Greek words "khroma" and "graphein" meaning "color" and "to write" or "to

represent". The chromatography technique is first discovered by Russian Biologists, Dr

Michael Tswett in 1906 for the separation of colored plant pigment on a column of alumina.

Now-a-days various types of chromatography are used to separate almost any given mixture

whether colored or colorless into its component.

Chromatography may be regarded as an analytical technique employed for the purification

and separation of organic and inorganic substances. It is also found useful for the

fractionation of complex mixture, separation of closely related compounds, such as isomers

and in the isolation of unstable substances.

Although there are several different types of chromatography, in each case a substance is

placed onto or into a medium and a solvent is passed through the test substance. In

chromatography science, the solvent is called "the mobile phase" or "the carrier fluid" and the

medium is called "the stationary phase".

Types of Chromatography:

There are three ways to classify chromatography. They are-

a) Classification of chromatography according to mobile phase:

1. Liquid chromatography: mobile phase is a liquid. (Liquid-Liquid

Chromatography, Liquid-Solid Chromatography).

2. Gas chromatography: mobile phase is a gas. (Gas-Solid Chromatography, Gas-

Liquid Chromatography)

b) Classification according to the packing of the stationary phase:

1. Thin layer chromatography (TLC): the stationary phase is a thin layer supported

on glass, plastic or aluminum plates.

2. Paper chromatography (PC): the stationary phase is a thin film of liquid supported

on an inert support.

3. Column chromatography (CC): stationary phase is packed in a glass column.


c) Classification according to the force of separation:

1. Adsorption chromatography.

2. Partition chromatography.

3. Ion exchange chromatography.

4. Gel filtration chromatography.

5. Affinity chromatography.

Table 01: Different Types of chromatography

MechanismMobile phaseStationary phaseMode or type

Solutes move at different rates

according to the forces of

attraction to the stationary

phase.

Liquid or gasSolid that attracts

the solutes

Adsorption

Chromatograph

y

Solutes equilibrate between the

2 phases according to their

partition coefficients

Liquid or gasThin film of

liquid formed on

the surface of a

solid inert

support

Partition

Chromatograph

y

Solute ions of charge opposite

to the fixed ions are attracted to

the resin by electrostatic forces

& replace the mobile counter-

ions.

Liquid

containing

electrolytes

Solid resin that

carries fixed ions

& mobile

counter-ions of

opposite charge

attached by

covalent bonds

Ion Exchange

Chromatograph

y

Molecules separate according to

their size:

LiquidPorous gel with

no attractive

Molecular

Exclusion


1. Smaller molecules enter the

pores of the gel, and need a

larger volume of eluent.

2. Larger molecules pass

through the column at a faster

rate.

action on solute

molecules

Chromatograph

y

Special kind of solute

molecules interact with those

immobilized on the stationary

phase

Liquid or gasSolid on which

specific

molecules are

immobilized

Affinity

Chromatograph

y

Applications of Chromatography:

Chromatographic methods will separate ionic species, inorganic or organic, and molecular

species ranging in size from the lightest and smallest, helium and hydrogen, to particulate

matter such as single cells. No single configuration will accomplish this, however. Little pre-

knowledge of the constituents of a mixture is required. At its best, chromatography will

separate several hundreds of components of unknown identity and unknown concentrations,

leaving the components unchanged. Amounts in the picogram or parts per billion ranges can

be detected with some detectors.

The solutes can range from polar to nonpolar— i.e., water-soluble to hydrocarbon-soluble.

Substances of low critical temperature or low molecular weight, such as the gases at

laboratory conditions showing dispersive or London intermolecular forces only, are separated

with molecular sieves or gas-solid techniques. Gas-liquid chromatography is applicable to

species with high critical temperatures and normal boiling points as high as 400° C.

Substances that are solids at normal laboratory conditions with molecular weights below1000

are best separated with liquid-solid or liquid-liquid systems. Lower members of the molecular

weight scale range are amenable to separations. Methods are involved at molecular weights

above 1,000. Field-flow fractionation extends the size range to colloids and microscopic

particles.


General uses of chromatography in our real life are:

Pharmaceutical Company – determine amount of each chemical found in new product.

Hospital – detect blood or alcohol levels in a patient’s blood stream.

Law Enforcement – to compare a sample found at a crime scene to samples from

suspects.

Environmental Agency – determine the level of pollutants in the water supply.

Manufacturing Plant – to purify a chemical needed to make a product.

Biotechnology industry – establishing the purity or concentration of compounds in

biotechnological research.

Biological application – Chromatography has many applications in biology. It is used to

separate and identify amino acids, carbohydrates, fatty acids, and other natural

substances. Environmental testing laboratories use chromatography to identify trace

quantities of contaminants such as PCBs in waste oil and pesticides such as DDT in

groundwater. It is also used to test drinking water and test air quality. Pharmaceutical

companies use chromatography to prepare quantities of extremely pure materials. The

food industry uses chromatography to detect contaminants such as aflatoxin.

For example we can consider the use of chromatography in Forensic Toxicology.

Forensic toxicology is the application of toxicology for the purpose of law.

Forensic Chemistry and Toxicology generally concerns the detection and characterization

of poisons or toxins exhibiting adverse physiological effects. Laboratory methods used in

chemical toxicological analysis cover a wide range and may be broadly classified as

follows: (1) physical tests (2) crystal tests (3) chemical spot tests (4) spectrophotometric

tests (5) chromatographic tests.

Toxicology is the study of substances that are harmful to human beings. Forensic

Toxicologists have the responsibility of detecting and identifying the presence of drugs

and poisons in fluids, tissues and organs. Their services are not just required in crime

laboratories and medical examiner's offices; they reach into hospital laboratories where


the identification of an overdose can mean the difference between life and death. The

work of a toxicologist generally falls into three main categories:

1. Routine testing for alcohol in blood or urine samples following a "breathalyzer" test.

However with the advent of new technology much of the testing can be carried out by

police with fewer cases being passed on to the forensic laboratory. Like Gas

chromatography was, and still is, used to identify exactly how much alcohol is present

in the blood or urine of a suspect. Because it is very accurate, results can be used as

evidence in a court of law.

2. Identification of drugs such as heroin, cocaine, cannabis, etc. It is common to perform

preliminary color or microcrystalline tests before using chromatography to identify a

substance as chromatography yields somewhat inconclusive results. However, both

thin layer and gas chromatography in conjunction with the preliminary tests is well

suited for drug analysis.

3. Detection of drugs and poisons in body fluids, tissues and organs. This area of

forensic toxicology involves the analysis of organs, tissues and body fluids in such

circumstances as sudden deaths and suspected poisonings. A post mortem is

performed by a pathologist who sends specimens of various body tissues and fluids to

the forensic toxicologist for examination. Many techniques are used in this area of

work including chromatography

Depend on the mechanisms different types of chromatography are specified in uses of

different actions. Applications according to the mechanism are given bellow:

Applications of Paper Chromatography:

1. Paper chromatography has widely been used for quantitative analysis of Inorganic,

organic and biochemical interest.

2. Paper chromatography is ideally suited for rapid analysis of reaction mixture and so it

is versatile tool in the hand of organic chemists.

3. Paper chromatography has been successfully used for characterizing and isolating the

following organic compounds.

Acids, Alcohols,


Glycols,

Alkaloids,

Amines,

Amino acids

Proteins and peptides,

Antibiotics etc.

4. Paper chromatography has also been used in the analysis of mixture of sugars.

5. It can be used to detect traces of pollutants in water food or in soil.

6. It can be used for the identification of compounds in drugs, in biochemical

preparation and in natural products. It can be used for checking the purity of samples.

Applications of Gel-filtration Chromatography

1. Analytical applications

Analytical group separation

Analytical fractionation

Determination of molecular masses: Determination of M. wt. of peptides, proteins

& polysaccharides

2. Preparative applications

Preparative fractionation

Preparative group separations

Separation of mixture of mono-and polysaccharides.

Separation of amino acids from peptides & proteins.

Separation of proteins of different molecular weights.

Separation of mucopolysaccharides & soluble RNA.

Separation of myoglobin & haemoglobin.

Separation of alkaloids & purification of enzymes.

3. Biochemical applications: In general, Gel-filtration chromatography which is also

known as Size Exclusion chromatography is considered a low resolution

chromatography as it does not discern similar species very well, and is therefore often


reserved for the final "polishing" step of purification. The technique can determine the

quaternary structure of purified proteins that have slow exchange times, since it can

be carried out under native solution conditions, preserving macromolecular

interactions.

Size Exclusion chromatography can also assay protein tertiary structure, as it

measures the hydrodynamic volume (not molecular weight), allowing folded and

unfolded versions of the same protein to be distinguished. For example, the apparent

hydrodynamic radius of a typical protein domain might be 14 Å and 36 Å for the

folded and unfolded forms, respectively. Size Exclusion chromatography allows the

separation of these two forms, as the folded form will elute much later due to its

smaller size.

4. Polymer synthesis: Gel-filtration chromatography or Size Exclusion chromatography

can be used as a measure of both the size and the polydispersity of a synthesized

polymer, that is, the ability to be able to find the distribution of the sizes of polymer

molecules. If standards of a known size are run previously, then a calibration curve

can be created to determine the sizes of polymer molecules of interest in the solvent

chosen for analysis. In alternative fashion, techniques such as light scattering and/or

viscometry can be used online with Size Exclusion chromatography to yield absolute

molecular weights that do not rely on calibration with standards of known molecular

weight. Due to the difference in size of two polymers with identical molecular

weights, the absolute determination methods are, in general, more desirable. A typical

Size Exclusion chromatography system can quickly (in about half an hour) give

polymer chemists information on the size and polydispersity of the sample. The

preparative Size Exclusion chromatography can be used for polymer fractionation on

an analytical scale.

Applications of Thin Layer Chromatography

1. As a check on process: It has been used for checking of the other separation

procedures and purification processes.

2. In Organic Chemistry:

The main use of Thin Layer Chromatography is isolation and separation of

individual components of a mixture.


The main reasons for popularity of Thin Layer Chromatography as an analytical and

preparation methods are:

It can be used for most of chemical compounds.

It has high speed of separation.

Its selectivity is high.

The following are the various applications of Thin Layer chromatography in organic

chemistry:

For checking the purity samples as a purification process.

Examination of reactions.

For identifying organic compounds.

Thin Layer Chromatography has been successfully used for characterizing and

isolating the following organic compounds.

i. Acids

ii. Alcohols

iii. Glycols

iv. Alkaloids

v. Amines

vi. Amino acids, proteins and

peptides

vii. Antibiotics

Besides these, there are compounds like carbohydrates, carbonyl compounds, Dyes,

Hydrocarbons, lipids, nucleic acids, pesticides, natural pigments, pharmaceutical products,

phenols, steroids, terpenes, essential oils, vitamins, adhesives, explosives plasticizers etc.

which have been separated and characterized by Thin Layer Chromatography.

3. For separation of Inorganic Ions:-

Recently Thin Layer Chromatography has been used for separating cationic,

anionic, purely covalent species and also some organic derivatives of the metals.

4. Applications of Thin Layer Chromatography in quantitative analysis:


i. Spectrophotometric

Measurement

ii. Fluorimetric Method

iii. Visual comparison of spots

iv. Spectral reflectance

v. Spot densitometer

vi. Vapour phase chromatography

vii. Radioactive methods

viii. Volumetric analysis

Application of Column Chromatography:-

1. Analytical uses: For analytical purposes, column chromatography finds limited

applications. Vestergaard and Sayegh could separate seven urinary steroids within 5

hours which requires 36 hours on a normal column. They have used narrow Teflon

tubing packed with aluminum oxide or silica gel.

2. Separation of geometrical isomers: The separation of cis/trans isomer is based on the

steric factors. Isomers whose functional groups can approach the surface of the

adsorbent more easily are more strongly adsorbed.

3. Separation of Diastereomers.

4. Separation of tautomeric mixtures.

5. Separation of racemates.

Applications of Affinity chromatography:

1. Purify and concentrate a substance from a mixture into a buffering solution.

2. Reduce the amount of a substance in a mixture.

3. Discern what biological compounds bind to a particular substance, such as drugs.

4. Purify and concentrate an enzyme solution.

Applications of Ion Exchange Chromatograph:

1. Water softening: Removal of Ca2+, Mg2+ & other multivalent ions causing hardness

of water by filtration through a layer of strong cation resin.

2. Separation of electrolytes from non-electrolytes.


3. Neutralization: Cationic exchanger in [H+] can be used to neutralize alkali hydroxide

& anionic exchanger in [OH-] form to neutralize the acidity.

4. Water demineralization: Removal of cations & anions dissolved in water. Usually

carried by the two step technique in which two columns of strongly acid cation

exchanger in [H+] form & strongly basic anion exchanger in [OH-] form are used in

sequence.

5. Separation of carbohydrates & their derivatives:

Uronic acids separated on anion exchanger.

Sugars converted into ionized form by using borate& separated on strong anion

exchanger.

Hexosamines separated on strong cation exchanger.

Medical Applications of High Performance Liquid Chromatography:

The isolation and purification of compounds is of critical importance to medicinal

chemists discovering and developing drugs. In these activities it is extremely important

to be able to produce mg quantities of the target compounds, from a synthetic reaction

mixture. Then later on use the same separation technologies to provide gram and Kilo

quantities. Similarly, the technique can isolate, purify and concentrate suitable

quantities of low level impurities and metabolites to support compound identification

and further characterization.

One of the key advantages of high performance counter current chromatography is that

it scales easily and simply. This allows the medicinal chemist to focus on the value-

added part of the development process i.e. the chemistry, and alleviates the need to

waste time developing scaled-up chromatography techniques, as demand for the

quantity of compound increases.

A further important issue is sample solubility since this can affect the throughput to

produce a specified quantity of the compound of interest. Typically, this can become an

issue when the purification is performed in reverse phase (RP) which generates aqueous

fractions. These aqueous fractions are laborious to concentrate by evaporation and this

process can lead to degradation of the product.


As scale of production increases the volumes of solvents used and those of product

containing fractions also increase. This is particularly an issue where Reverse Phase-

High Performance Liquid Chromatography produces large volumes of aqueous

fractions. High performance counter current chromatography can be used in normal

phase for the same separation which means that fractions can be collected in essentially

non-aqueous solvents which makes their processing simpler, faster and less energy

consuming.

High Performance Liquid Chromatography has found many applications in medicinal

chemistry, to which the Spectrum or Midi bench top products are normally found to be

the most suitable for the quantities of compound required.

Applications of Chromatography

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